1. Field of the Invention
This invention relates generally to the field of nucleic acid probe technology. More specifically, it relates to new compositions and methods to identify and quantify targeted DNA or RNA sequences, including single-base sequence variants in genomic and pathogenic samples. In particular it relates to new general probe systems to detect and assess target amplification by real-time PCR (polymerase chain reaction) with improved sensitivity, quantification and economy.
2. Description of the Related Art
The detection of targeted polynucleotide sequences is usually based on the use of short synthetic oligonucleotide probes or long cDNA based probes that are labeled and hybridized to a target sequence of interest. To work effectively, such probe products must be washed after hybridization to remove both unbound probes and probes that are weakly bound to non-specific targets. However, under the conditions of real-time PCR [U.S. Pat. No. 4,965,188; U.S. Pat. No. 5,210,015; U.S. Pat. No. 5,487,972; U.S. Pat. No. 5,538,848], a wash step is not feasible, and thus novel probes had to be devised that only generate signaling when they are bound to a complementary target and that have diminished or quenched signaling when they are unbound and floating free in solution.
To achieve this end, the prior art has generally relied on probes that employ FRET (fluorescent resonance energy transfer) interactions between a donor and an acceptor molecule, such as two fluorophores or a fluorophore and a quencher [Didenko V, Biotechniques, 2001, November; 31(5):1106-16, 1118, 1120-1121; Chen et al., Proc. Natl. Acad. Sci. USA, 1997, Sep. 30; 94: 10756-10762]. To work, the fluorescence emission spectrum of the donor must overlap the absorption or excitation spectrum of the acceptor.
In FRET, the excited-state energy of the fluorescent donor molecule is transferred to the acceptor molecule when they are brought into proximity (10 to 100 angstroms). If the acceptor molecule is fluorescent, signaling shifts to a longer wavelength. If the acceptor molecule is an effective quencher, fluorescent signaling is significantly diminished and may be essentially turned off. Taqman and Molecular Beacon probes are the principal FRET-based probes of this type for real-time PCR detection. In both cases, they serve as an internal probe that is used in conjunction with a pair of opposing primers that flank the target region of interest. When the primers amplify the target segment, the probe will selectively bind to those products at an identifying sequence in between the primer sites, thereby causing increases in FRET signaling relative to increases in target frequency. While these probe systems are therefore similar in effect, they employ somewhat different detection mechanisms.
More specifically, a Taqman probe comprises a synthetic oligonucleotide of about 22 to 30 bases that is complementary to a target sequence and that is labeled on both ends with a FRET pair [Livak et al. 1996, U.S. Pat. No. 5,538,848]. Typically, the 5′ end will have a shorter wavelength fluorophore such as fluorescein (e.g. FAM™) and the 3′ end is commonly labeled with a longer wavelength fluorescent quencher (e.g. TAMRA™) or a non-fluorescent quencher compound (e.g. Black Hole Quencher™). In solution, the probe will randomly coil or fold so that the labeled ends are in proximity and 5′ fluorescent emissions are effectively quenched. But when the probe binds to an internal target sequence during the annealing step of PCR, the advancing Taq polymerase has 5′-3′ exonuclease activity that will degrade the bound probe, permanently releasing the components in solution. Once a 5′ fluorophore is thereby released, it can emit fluorescent signaling, and thus the level of fluorescence that results is proportional to the frequency of amplified targets.
Taqman probes evolved from an earlier system based on 5′-3′ exonuclease digestion using internal probes with simply a 5′ fluorescent end. When the probe was digested via polymerase advance, the released fluorescent fragments were thereafter detected [U.S. Pat. No. 5,210,015; U.S. Pat. No. 5,487,972]. One common requirement for designing such fluorescent probes is that there be no guanine (G) at the 5′ end since a G adjacent to the reporter dye will significantly quench reporter fluorescence even after the probe is degraded.
Like TaqMan probes, Molecular Beacon probes use FRET interactions to detect and quantify a PCR product, with each probe having a 5′ fluorescent-labeled end and a 3′ quencher-labeled end. [Tyagi et al. 1999, U.S. Pat. No. 5,925,517; Tyagi et al., Nature Biotechnology 1996, 14: 303-8]. However, Molecular Beacons also include short artificial segments of 5 to 7 bases at each end that are complementary to one another but not complementary to the target. In the absence of target binding, these matching end sequences will bind together in solution, thereby bringing the quencher-labeled end in proximity to the fluorophore-labeled end so that fluorescent signaling is suppressed.
This probe structure has been described as a hairpin or stem-loop configuration, wherein the stem depicts the two short self-binding ends and the loop depicts the long internal target-specific region of about 20 to 30 bases. Due to this configuration and the relatively greater length of the target-specific region, these probes will preferentially hybridize to available complementary targets, thereby causing the probes to straighten and extend since double-stranded DNA is relatively rigid like a spring as compared to single-stranded DNA which is floppy and easily folded like a string. Consequently, with target binding, the labeled ends of the probe will separate from one another, thereby releasing fluorescent emissions. Because this mechanism does not depend on degradation of the probe, Molecular beacons can be employed in a variety of detection schemes in addition to real-time PCR assays. A similar detection mechanism is employed by an independent invention wherein the probe is fabricated with only the loop structure and without the short complementary stem regions [Mayrand et al. 1997, U.S. Pat. No. 5,691,146]. This method also works because in the absence of target binding the fluor-labeled and quencher-labeled ends will naturally fold or coil together to significantly quench fluorescence, whereas, when target binding occurs, these probes will automatically stretch out, thereby releasing fluorescence.
An alternative FRET-based approach for PCR and real-time PCR detection uses two hybridization probes that bind to adjacent sites on the target wherein the first probe has a fluorescent donor label at the 3′ end and the second probe has a fluorescent acceptor label at its 5′ end [Wittmer et al. Biotechniques, 1997, 22: 130-138]. When both probes bind to the template, bringing the donor and acceptor labels into proximity, the FRET interaction occurs causing a color shift in fluorescent signaling. Thus PCR amplification causes an exponential increase in the ratio of acceptor fluorescence versus donor fluorescence which is proportional to the amount of target DNA generated.
Scorpion™ probes provide a FRET-based stem-loop detection mechanism similar to Molecular Beacons, except that the probe also has a segment attached that serves as an amplification primer [Whitcombe et al. Nat Biotechnol. 1999, Aug. 17(8): 804-7; U.S. Pat. No. 6,326,145]. Like Molecular Beacons, these probes maintain a stem-loop configuration in the unhybridized state with the fluorophore thereby quenched. However, they have a longer multi-component structure. First there is a 5′ fluorophore, then a target-specific stem-loop section, then a Black Hole Quencher™, then a hexethylene glycol (HEG) blocker and finally a 3′ primer sequence. The blocker prevents reverse extension of the product onto the probe. After primer extension occurs, the Scorpion probe is attached to the terminal end of the amplicon. When denaturation occurs again, followed by annealing, the loop segment of the probe will preferentially bind to its long complementary segment on the attached template, thereby opening the stem-loop structure and releasing fluorescence.
Alternatively, the stem-loop structure is cut into two units with one unit having four components, i.e., the 5′ fluorophore, the target specific segment, the blocker and the primer, and with the other unit having the quencher and a probe segment. Similar to Scorpion probes, Sunrise™ probes comprise a primer attached to a hairpin probe that is extended during amplification. This separates the internal quencher label from the 5′ terminal fluorophore [Nazarenko et al., Nucl. Acids Res. 1997, 25: 2516-2521].
All these dual-labeled FRET-based probes require careful design and they are quite expensive. Their synthesis is difficult and they require manual post-synthesis addition of at least one label as well as high pressure liquid chromatography to purify for double-labeled products. Taqman and Molecular Beacon probes also require the design of two opposing primers that must work in conjunction with the probe. In order to function effectively during the annealing step, Taqman and Molecular Beacon probes must be longer and have a Tm that is 5 to 10 degrees higher than the primers since the probe must bind firmly to the target before extension. For Taqman probes this condition is needed to digest and release fluorescence. For Molecular Beacons, this condition is similarly essential to stretch out and release fluorescence. However, at the same time, the requirement for a long target-specific probe makes it much more difficult to design and develop probes that can selectively detect small sequence changes such as SNPs (single nucleotide polymorphisms) or single base mutations. Therefore, these hybridization-based probes generally require multiple designs and repeated testing in order to achieve a working or optimal result.
In other detection formats, different issues are important. When gene targets are detected by FISH (fluorescent in situ hybridization) four processing steps are typically required: 1) the preparation of labeled probes, 2) probe hybridization to fixed denatured targets, 3) the washing of unbound probes, and 4) fluorescent excitation and detection [Barch M J, editor. The ACT Cytogenetics Laboratory Manual. 2nd ed. New York: Raven Press; 1991]. Careful wash steps are critical to effective detection since the signal to noise ratio is highly dependent on the stringency of washing and since excessive washing can greatly reduce signaling. Thus FISH probes generally require a delicate, graded series of critically timed wash steps, using different components and dilutions, all of which add considerably to the time, complexity and cost of such assays.
Microarray detection is somewhat similar to FISH detection. Arrays are typically based on printing glass or silicon substrates with bound oligo probes or cDNA probes; applying fluorescent-labeled DNA or RNA targets which must be hybridized to the probes; washing the arrays stringently; and then detecting the bound targets—usually by laser scanning [Schena et al. 1995, Science 270: 467-470; Heller et al. 1997, P.N.A.S. USA, 94, 2150-2155]. Like FISH probes, the wash steps are again complex and time consuming. However, the preparation and labeling of the targets are an expensive additional process since each target sample is unique. Indeed, the cost and time burden of current target labeling and detection methods is the major limitation to the routine use of microarray-based assays.
The present invention significantly overcomes many of the limitations described above and provides advantages over the prior art for microarray detection, FISH detection and PCR detection. In addition, several embodiments of the present invention enable more definitive quantification of real-time PCR products, whereas such assays typically require running concurrent standards and controls in order to certify results.
The invention is especially directed towards the detection of single base variants important to discriminating bacterial and viral pathogens, including drug resistant mutants. Of particular interest, HIV-1 resistance-related targets have defied detection with ordinary real-time PCR probe systems or with hybridization-based microarray probes because these critical single base mutations commonly occur in a sea of nearby unrelated mutations [Shafer, R. W. 2002, Clin Microbiol Rev 15: 247-277; D'Aquila et al. 2002, Topics HIV Med 10: 21-25; Gonzalez et al. 2004, Journal of Clinical Microbiology, 42 (7): 2907-2912; Gunthard et al. 1998, AIDS Res Hum Retroviruses 14: 869-876]. Overcoming these difficult detection limitations has been a goal as well as an accomplishment of the present invention.
There is a recognized need in the art for improved detection of nucleic acid targets. Specifically, the prior art is deficient in the lack of a simple method for detecting single or multiple polynucleotide target sequences by employing novel probe and antiprobe compositions and detection strategies in various liquid and solid phase detection platforms. The present invention fulfils this longstanding need in the art.